Project Summary Dendritic cells are the sentinels of the immune system. They patrol the body looking for antigens and then migrate to a lymph node to communicate what they found to T cells and other cells of the adaptive immune system. These professional migrators and searchers are a critical component of human immunity, and their migration is targeted or hijacked by multiple pathogens including some pox and herpes viruses, tuberculosis, and anthrax. Since dendritic cells can activate cytotoxic T cells to attack cancer cells, their migration also plays a role in cancer immunotherapy strategies. Many cells, including dendritic cells, migrate by extending lamellipodia. Lamellipodia are thin, planar protrusions that have been extensively studied for cells migrating on 2D surfaces, such as glass coverslips. Dendritic cells use lamellipodia to find a path through crowded 3D environments and to enter lymphatic vessels. Lamellipodia and the actin network that composes them have been studied for decades. However, most molecularly detailed models of lamellipodia regulation and function were derived from studying cells on 2D surfaces, so we still do not know how cells initiate and extend lamellipodia in 3D environments. Dr. Driscoll will investigate how actin nucleators organize to generate sheet- like lamellipodial morphologies in the absence of a surface to guide their generation, as well as how actin nucleators organize to direct the extension of lamellipodia within crowded 3D environments. As a model of dendritic cell migration through peripheral tissues, she will study their migration though 3D fibrous collagen matrices. Widely available microscopic techniques, such as confocal microscopy, cannot image cells in 3D collagen with the spatial and temporal resolution required to measure the organization of actin nucleators in lamellipodia. However, recently developed techniques, such as light-sheet microscopy, are just beginning to be able to do so. Since light-sheet microscopes can easily produce 3D movies exceeding 1TB in size, interpreting and even simply visualizing such large amounts of data requires sophisticated computing workflows. Although Dr. Driscoll has recently developed computational tools to analyze light-sheet microscopy images, to utilize these tools she needs further training in building and using light-sheet microscopes. The Danuser and Fiolka labs at UT Southwestern are ideal locations to obtain this training. Dr. Gaudenz Danuser is an expert at developing computer vision tools to address cell biology questions, whereas Dr. Reto Fiolka is specialized in light-sheet microscopy of 3D systems. The training Dr. Driscoll receives will enable her to lead an independent laboratory that focuses on how cells migrate through and interact with their 3D environment. In summary, Dr. Driscoll will integrate light-sheet microscopy, 3D image analysis, and molecular biology techniques to determine how lamellipodia form and function in 3D environments.